This invention relates to methods for the production of chiral compounds, and in particular to methods for the production of chiral hydroxy-amino compounds. The hydroxy-amino compounds have applications in the synthesis of pharmaceutical products.
Natural and non-natural xcex1-hydroxy-xcex2-amino acids and xcex2-hydroxy-xcex3-amino acids and their derivatives occur in many biologically active natural products and are important intermediates in the synthesis of various pharmaceuticals. One of the most important xcex1-hydroxy-xcex2-amino acids is the side chain of the potent anticancer drug Taxol. Various derivatives of this xcex2-amino acid have been synthesized and linked to the polycyclic core ring of Taxol in an effort to improve the potency and the spectrum of uses of this important drug.
The xcex2-hydroxy-xcex3-amino acid structural motif is encountered in a number of natural products and current and developmental drugs. Some of the most common xcex2-hydroxy-xcex3-amino acids include statine, isostatine and benzyl statine (phenylstatine) (FIG. 1). Statine is the key component of pepstatin, a naturally-occurring hexapeptide antibiotic, which acts as an inhibitor of aspartic acid proteases such as rennin, pepsin and cathepsin D [Umezawa, H etal J. Antibiotics 23, 259 (1970); Ric, D. H. J. Med. Chem 23, 27 (1980)]. The low selectivity of pepstatine has led to the development of more specific synthetic analogues by substituting the isobutyl moiety of statine with more lipophilic substituents such as cyclohexylmethyl, which led to the widely used analogue cyclohexyl-statine. Isostatine is an essential amino acid in Didemnins [Sakai, R. at al J. Am. Chem. Soc. 117, 3734 (1995); Joullie, M. M. J. Am. Chem. Soc 112, 7659 (1990)], a group of cyclic peptides which show strong antitumor, antiviral, and immunosuppressive activity (Sakai, R. et al. J. Med. Chem. 39, 2819 (1996)]. Benzyl statine is part of the biologically active compounds hapalosin (Stratmann, K et al J. Org. Chem. 59, 7219 (1994); Armstrong, R. W. J. Org. Chem. 60, 8118, (1995)] and dolastatin 10 [Shiori, T et al Tetrahedron 49, 1913 (1993)]. In particular hapalosin restores the lethal activity of cytotoxic antitumor drugs (such as actinomycin D, colchicines and taxol) to cancer cells by breaking the P-glycoprotein-mediated multi-drug resistance caused by the export of the cancer drugs from the cell using transmembrane P-glycoproteins. 
FIG. 1: Natural products and related pharmaceuticals that contain xcex3-amino-xcex2-hydroxy amino acids.
Other xcex2-hydroxy-xcex3-amino acids that are incorporated in molecules with biological activities include (2S,3S,4R)-4-amino-3-hydroxy-2-methyl pentanoic acid, which is the amino acid linker of bleomycin B2 and the main constituent of the powerful carcinostatic blenoxane [Boger, D. L et al J. Am Chem Soc 116, 5607, (1994)] and (2R,3S,4R)-4-amino-3-hydroxy-2-methyl-5-(2xe2x80x2-pyridil) pentanoic acid, which is part of pyridomycin [Kinoshita, M; Awamura, M. Bull. Chem. Soc 51, 869 (1978)], a Streptomyces-synthesized anti-mycobacterial drug (FIG. 2). Statines and related compounds based on xcex2-hydroxy-xcex3-amino acids are particularly prevalent in anti-cancer drugs and drug candidates. The absolute stereochemistry of these molecules is important for biological activity. 
FIG. 2: Potent biologically-active natural products that contain xcex3-amino-xcex2-hydroxy amino acids.
Another important motif in pharmaceutically-active compounds is the xcex1-hydroxy-xcex2-amino acid structural unit. Among the examples of pharmaceutical products that contain the xcex1-hydroxy-xcex2-amino acid moiety as a key component in their structures are molecules such as bestatin, amastatin and ubenimex, which possess immunoregulatory, antitumor and antimicrobial activities. The ability to prepare compounds in this class with defined absolute stereochemistry is critical to the commercial synthesis of these compounds and their analogs.
Despite the general importance of hydroxyl-substituted xcex2- and xcex3-amino acids and their derivatives as pharmaceutical intermediates, the preparation of these compounds remains a significant challenge to chemists. Most of the synthetic approaches toward the production of xcex1-hydroxy-xcex2-amino acids are purely chemical transformations that require multi-step reaction sequences, chiral catalysts or starting materials, and stringent or air-sensitive reaction conditions. Occasionally the synthetic methods involve the production of relatively unstable intermediates. Most of the chemical syntheses of statine and isostatine, for example, begin from the natural xcex1-amino acids leucine and isoleucine, respectively [Hamada, Y. et al J. Am Chem Soc, 111, 669 (1989); Tao, J.; Hoffmann, R. V J. Org. Chem 62, 2292 (1997)]. After protection of the amino group (PG=protecting group), an aldol or Claisen condensation to the xcex2-keto-xcex3-amino acid followed by a reduction gives the desired xcex2-hydroxy xcex3-amino acid product (FIG. 3). 
FIG. 3: Outline of the most common current chemical syntheses of xcex2-hydroxy-xcex3-amino acids.
Some of the problems encountered in these syntheses are the isomerization of the xcex3-carbon under the basic conditions of the condensation reaction, the many steps required (often 7-10), and the low diastereoselectivity of the final reduction step, which often times gives the wrong diastereomer as the major product [Kessler, H; Schudok, M Synthesis 457 (1990); Maibaum, J.; Rich, D. H J. Org. Chem 53, 869 (1988)]. An obvious drawback in using methods based on natural amino acid precursors for the synthesis of xcex2-hydroxy-xcex3-amino acids is that non-natural xcex1-amino acid counterparts cannot always be easily accessed, and for this reason other chemical synthetic schemes have been developed. The xcex2,xcex3-amino alcohol moiety in one alternative synthetic route is synthesized from xcex1,xcex2-unsaturated alcohols that are epoxidized using a chiral catalyst, followed by a ring opening using an nitrogen nucleophile (FIG. 3) [Catasus, M. et al Tetrahedron Lett 40, 9309 (1999); Catejon, P. et al Tetrahedron 52, 7063. (1996)]. Although good enantiomeric purity of the product was reported (90-99% ee), this methodology is long (6-10 steps), gives moderate yields (20-40%), and requires expensive catalysts and stringent air-sensitive reaction conditions. Other methods for synthesizing xcex2-hydroxy-xcex3-amino acids involve Wittig reactions of chiral oxazolidinones [Reddy, G. V et al Tetrahedron Lett 40, 775 (1999)] asymmetric Claisen rearrangements [Krebs, A.; Kazmaier, U. Tetrahedron Lett. 40, 479 (1999)], selective Grignard reaction of N-protected amino acids [Veeresha, G.; Datta, A Tetrahedron Lett 38, 5223 (1997)] or the use of doubly chiral precursors [Kwon and Ko, Tetrahedron Lett 43, 639-641 (2002)]. Again, long and complicated reaction sequences and chiral starting materials and/or catalysts are required using these methodologies.
Enzyme catalysis offers an alternative to purely chemical synthetic schemes. Enzymatic methods that have been reported to date are resolutions of a racemic mixture, having a maximum yield of 50% for the resolution step alone. Challenges similar to those encountered in the chemical synthesis of xcex2-hydroxy-xcex3-amino acids are also faced in the chemical synthesis of xcex1-hydroxy-xcex2-amino acids. In both cases, gaining control over the stereochemistry of the chiral carbons bearing both the amino and the alcohol groups at reasonable cost and high enantiomeric purity is the key to the successful production of these important chemical intermediates.
Chiral hydroxy compounds can be produced by the stereoselective reduction of ketones catalyzed by ketoreductase enzymes. As used herein, the term ketoreductase means any enzyme that catalyzes the reduction of a ketone to form the corresponding alcohol. Ketoreductase enzymes include those classified under the Enzyme Commission numbers of 1.1.1. Such enzymes are given various names in addition to ketoreductase, including, but not limited, to alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, hydroxyisocaproate dehydrogenase, xcex2-hydroxybutyrate dehydrogenase, steroid dehydrogenase, sorbitol dehydrogenase, aldoreductase, and the like.
Many examples of enzymatic reductions of various classes of substrates have been reported [Wong, C-H; Whitsides, G. M. Enzymes in Synthetic Organic Chemistry, Pergamon, N.Y., (1994); Sugai, T Curr. Org. Chem 3, 373 (1999)]. Various alcohol dehydrogenases have been investigated [Patel, R. N Adv. Appl. Microbiol 43, 91 (1997); Riva, S.; Carrea, G. Angew. Chem. Int. Ed 39, 2226 (2000)]. A well known example is horse liver alcohol dehydrogenase (HLADH), an enzyme that has been very extensively studied and can reduce aldehydes and ketones to the corresponding alcohols, in some cases providing alcohols in good enantiomeric purity. The substrate range is limited and does not include most xcex2-ketoesters, however.
Various ketoreductase enzymes have been identified that catalyze the stereoselective reduction of a range of different ketones, including xcex2-ketoesters. [See, for example, J. David Rozzell, ACS Symposium Series 776, Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals, B. C. Saha and D. C. Demirjian, eds., pp.191-199, (2000) and references therein, all hereby incorporated by reference.] These enzymes have been shown to act on a number of structurally diverse ketones. The genes expressing a number of these broad-range ketoreductases have been cloned and expressed, and a number of these enzymes are readily available commercially (BioCatalytics, Inc, Pasadena, Calif. USA). In many cases, enzymes can be identified that can produce either stereoisomer of a chiral alcohol by stereoselective reduction of a target ketone. For example, when the Ketoreductase Screening Set (Catalog number KRED-8000, BioCatalytics, Inc, Pasadena, Calif. USA) containing 8 different ketoreductases was screened against either alpha-chloroacetophenone or ethyl 4-chloroacetoacetate, some enzymes could be found within the set that were R-selective while others were found that were S-selective with respect to the chiral alcohol produced.
It has also been demonstrated that ketoreductase enzymes can be used to catalyze the reduction of 2-substituted-3-ketoesters. The products of these reductions are compounds with two chiral centers, and depending on the enzyme employed, the reduction can be diastercoselective, as shown in FIG. 4. Such reactions have been described using isolated enzymes and with whole cells. When the enzymes within the Ketoreductase Screening Set (Catalog number KRED-8000, BioCatalytics, Inc, Pasadena, Calif. USA) were studied for the reduction of 2-ethyl-3-ketobutyrate ethyl ester, certain enzymes were shown to be highly diastereoselective for the reduction to the corresponding alcohol. [For other examples, see S. Rodriguez et al., J. Org. Chem., 65, 2586 (2000); S. Rodriguez et al., J. Am. Chem. Soc., 123, 1547 (2001) and references therein, hereby incorporated by reference.]
In contrast to the 2-substituted-3-ketoesters shown in FIG. 4, there is only a single report of the diastereoselective reduction of a xcex2-ketodiester such as that depicted in FIG. 5. 
Benner and coworkers used actively fermenting Baker""s yeast to carry out the reduction of the compounds shown in FIG. 5 where n is 1 and R is allyl or propargyl [T. Arsian and S. A. Benner, J. Org. Chem., 58, 2260-2264 (1993) and references therein, hereby incorporated by reference]. These compounds were prepared as potential precursors for the synthesis of non-standard nucleic acid bases. These were the only compounds for which reduction with fermenting yeast was reported, and the ketoreductase or ketoreductases involved were neither isolated nor determined. The reduction reaction was reported to be enantioselective and diastereoselective, although the degree of selectivity observed varied widely depending on reaction conditions, and yields in some cases were diminished by the partial metabolism of the substrate.
There are no reports of the highly diastereoselective reduction of a range of substituted xcex2-ketodiesters, nor any reports of the use of substituted xcex2-ketodiesters in the production of xcex1-hydroxy-xcex2-amino acids and xcex2-hydroxy-xcex3-amino acids using a reaction sequence incorporating a diasereoselective reduction of substituted xcex2-ketodiesters.
The present invention is directed toward methods for the production of hydroxy-amino acids in general, and to the production of xcex1-hydroxy-xcex2-amino acids and xcex2-hydroxy-xcex3-amino acids in particular. The methods of the present invention are broadly applicable for the synthesis of a wide range of chiral hydroxy xcex2- and xcex3-amino acids from inexpensive and easily accessible starting materials.
In one embodiment, the invention is directed to a method for producing a hydroxy-amino acid or a derivative thereof. A substituted xcex2-ketodiester having a ketone group and two ester functional groups is contacted with a ketoreductase under conditions permitting the reduction of the ketone group to an alcohol. Only one of the ester functional groups is regioselectively hydrolyzed to the corresponding carboxylic acid, whereby a non-hydrolyzed ester functional group remains. Either the carboxylic acid or the non-hydrolyzed ester functional group is converted to an amine or a derivative thereof to produce a hydroxy-amino acid or derivative thereof.
A key step in the preparation of the target compounds is the diastereoselective reduction of substituted xcex2-ketodiesters to form the corresponding substituted hydroxydiesters. In one embodiment, the method of the present invention uses a reaction sequence comprising a diastereoselective enzyme-catalyzed reduction of a xcex2-ketodiester to introduce two or more chiral centers in a single step, followed by regioselective hydrolysis of only one of the two ester functional groups to form the corresponding carboxylic acid, and a conversion including a stereospecific chemical rearrangement in which the carboxylic acid is converted to an amine, or derivative thereof, to generate the desired hydroxy-amino acid, or derivative thereof In another embodiment, the method of the present invention uses a reaction sequence comprising a diastereoselective enzyme-catalyzed reduction of a xcex2-ketodiester, followed by regioselective hydrolysis of only one of the two ester functional groups to form the corresponding carboxylic acid, the conversion of the non-hydrolyzed ester functional group to an amide, a hydrazide, or a hydroxamic acid derivative, and a stereospecific chemical rearrangement in which the amide, hydrazide, or hydroxamic acid derivative is converted to an amine, or derivative thereof, to generate the desired hydroxy-amino acid, or derivative thereof. As used herein, as it pertains to a hydroxyamino compound, the words xe2x80x9cderivative thereofxe2x80x9d means a carbamate or urethane, which can be cyclic or acyclic, a urea, a hydrazide, or an amide formed from the amino group, or any protected form of the alcohol, including ethers, silyl ethers, alkyl esters, aryl esters, aralkyl esters, or carbonate esters.
In connection with this invention, it has been discovered that, when contacted with an appropriate ketoreductase, a broad range of substituted xcex2-ketodiesters, such as 3-substituted-oxaloacetic diesters (n=0 in FIG. 5) and 2-substituted-3-ketoglutarate diesters (n=1 in FIG. 5), can be reduced diastereoselectively as shown in FIG. 5, producing a substituted xcex2-hydroxydiester with 2 chiral centers. Preferably, the reaction catalyzed by the ketoreductase is substantially diastereoselective. As used herein, the term xe2x80x9csubstantially diastereoselectivexe2x80x9d means a reaction that produces a compound containing two or more chiral centers with the product mixture containing at least about 50% of a single diastereomer of the possible diastereoisomers, preferably at least about 75% of a single diastereomer, and more preferably at least about 90% of a single diastereomer. As a chiral synthesis rather than a resolution, yields up to 100% of theoretical can be achieved during the enzymatic reductions, with two chiral centers being introduced in a single step in a substantially diastereoselective manner. The diastereomeric hydroxy diesters can be further converted to xcex1-hydroxy-xcex2-amino acids and xcex2-hydroxy-xcex3-amino acids by regioselective hydrolysis of only one of the two ester functional groups and conversion of the remaining ester or carboxylic acid to an amine by a stereospecific rearrangement reaction which preserves the asymmetry at both chiral centers.
In a particularly preferred embodiment, the xcex2-ketodiester is a compound having the following formula: 
wherein n is 0 or 1, and R1, R2 and R3 are each independently selected is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclic, and substituted heterocyclic.
As used herein, the term xe2x80x9calkyl,xe2x80x9d alone or in combination, means a straight-chain or branched-chain hydrocarbon, either saturated or unsaturated, containing from 1 to about 12 carbon atoms. xe2x80x9cSubstituted alkylxe2x80x9d means alkyl groups substituted on one or more carbon atoms with one or more substituents selected from the group consisting of hydroxy, alkoxy, thio, thioalkyl, fluoro, chloro, bromo, iodo, carboxy, carboxyalkyl, carbamoyl, carbamide, amino, amidino, phosphate, phosphonate, phosphinate, phosphinyl, their derivatives, and the like.
As used herein, the term xe2x80x9caryl,xe2x80x9d alone or in combination, means a carbocyclic aromatic system containing from 1 to 4 rings, wherein said rings may be attached in a pendant manner to each other or may be fused to each other. Examples of aryl groups include phenyl, naphthyl, biphenyl, anthracenyl, and the like. Substituted aryl means aryl groups substituted on one or more carbon atoms with one or more substituents selected from the group consisting of hydroxy, alkoxy, thio, thioalkyl, fluoro, chloro, bromo, iodo, carboxy, carboxyalkyl, carbamoyl, carbamide, amino, amidino, phosphate, phosphonate, phosphinate, phosphinyl, their derivatives, and the like.
As used herein, the term xe2x80x9caralkylxe2x80x9d means an alkyl group as defined above substituted with an aryl group as defined above. Substituted aralkyl means aralkyl groups substituted on one or more carbon atoms with one or more substituents selected from the group consisting of hydroxy, alkoxy, thio, thioalkyl, fluoro, chloro, bromo, iodo, carboxy, carboxyalkyl, carbamoyl, carbamide, amino, amidino, phosphate, phosphonate, phosphinate, phosphinyl, their derivatives, and the like.
As used herein, the term xe2x80x9cheterocyclic,xe2x80x9d alone or in combination, means a saturated or unsaturated monocyclic or multi-cyclic group containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, sulfur, phosphorous, selenium, and silicon. Substituted heterocyclic means heterocyclic groups substituted on one or more carbon atoms with one or more substituents selected from the group consisting of hydroxy, alkoxy, thio, thioalkyl, fluoro, chloro, bromo, iodo, carboxy, carboxyalkyl, carbamoyl, carbamide, amino, amidino, phosphate, phosphonate, phosphinate, phosphinyl, their derivatives, and the like.
Some ketoreductase enzymes particularly useful in the present invention require the presence of nicotinamide cofactors in order to catalyze the reduction of the subject xcex2-ketodiesters. As used herein, the term xe2x80x9cnicotinamide cofactorsxe2x80x9d includes NAD+, NADH, NADP+, NADPH, and any derivatives thereof that can be used as cofactors by oxidoreductase enzymes. Nicotinamide cofactors useful in the present invention are readily available commercially from vendors, including Sigma-Aldrich Chemical Company (St. Louis, Mo. USA), BioCatalytics, Inc., (Pasadena, Calif. USA), Roche Diagnostics (Indianapolis, Ind. USA) and others well known to those skilled in the art. Derivatives of nicotinamide cofactors useful in the practice of this invention include the nicotinamide analogs reported in U.S. Pat. No. 5,801,006, the disclosure of which is hereby incorporated by reference, polyethyleneglycol functionalized nicotinamide molecules such as reported by Okada and Urabe in Methods in Enzymology, 136, 34-45 (1987), and the like. The concentration of nicotinamide cofactor used in the reaction mixture with a ketoreductase enzyme preferably ranges from about 0.001 mM to about 10 mM, and more preferably from about 0.01 mM to about 0.5 mM. For the stereoselective reduction to be carried out as described in the present invention, the reduced form of the nicotinamide cofactor (NADH, NADPH or analog thereof) is used by the ketoreductase enzyme. It is also possible to start with an oxidized form of the nicotinamide cofactor (NAD+, NADP+, or analog thereof), which is less expensive that the reduced form, provided that a source of reducing equivalents is furnished to reduce the oxidized form of said cofactor to the reduced form for the enzyme-catalyzed reduction to proceed.
In the method of the present invention, the nicotinamide cofactors can be recycled, if desired. Cofactor recycling can be achieved in cell-free enzymatic reactions by the use of an appropriate recycling enzyme in combination with a ketoreductase. Enzymes useful for the recycling of nicotinamide cofactors are well-known in the art, and include formate dehydrogenases, glucose dehydrogenases, sorbitol dehydrogenases, alcohol dehydrogenases and the like. Any of the recycling methods known in the art may be used in the practice of this invention. Some examples of cofactor recycling methods are described in Preparative Biotransformations (S. M. Roberts, editor), 3.1.1-3.1.6, John Wiley and Sons, Chichester, U.K. (1996) and references therein; Z. Shaked and G. M. Whitesides, J. Am. Chem. Soc. 102, 7104-5 (1980) and references therein; J. B. Jones and T. Takamura, Can. J. Chem. 62, 77 (1984); all hereby incorporated by reference.
In the practice of this invention, cofactor recycling may also be achieved by the use of a microorganism into which the genes encoding both the ketoreductase and the recycling enzyme have been cloned and expressed together. In this embodiment, the whole cell may be used as the catalyst, or, if desired, the ketoreductase and the recycling enzymes may be isolated from the cell. As used herein with respect to enzymes, the term xe2x80x9cisolatedxe2x80x9d means extracted from or separated from cells. An isolated enzyme or enzymes may be used as a crude cell lysate, partially purified enzyme preparation, or a purified enzyme preparation.
In accordance with this invention, the ketoreductase and the recycling enzyme may be used as soluble enzymes or, if desired, one or both enzymes may be immobilized prior to use. When used as soluble enzymes, ketoreductases and recycling enzymes useful in the practice of this invention may be isolated from cells capable of producing the desired enzymes and used without purification, or purified partially or completely. The purification of the enzymes may be accomplished by techniques well known to those skilled in the art. Some examples of purification methods for enzymes are described in Methods in Enzymology, 22 (1971) and references therein, hereby incorporated by reference.
If the ketoreductase and the recycling enzymes are to be immobilized, techniques well known in the art can be used. Either the ketoreductase enzyme or the cofactor recycling enzyme may be immobilized separately, or both enzymes may be immobilized together. Such immobilization of the enzymes can be carried out by co-immobilization of both enzymes together on the same support material, or the ketoreductase and the recycling enzyme may be immobilized separately and the two immobilized enzymes can be combined in appropriate amounts for carrying out the diastereoselective reduction reaction. The appropriate amounts of immobilized enzymes to be used can be readily determined by persons skilled in the art. Methods for the immobilization of enzymes are well known to those skilled in the art. One example of an immobilized enzyme method useful in the practice of this invention is described by Weetall et al., Methods in Enzymology 34, 59-72 (1974), which is hereby incorporated by reference. In this method, enzymes may be immobilized on an amine-functionalized porous glass or ceramic support which has been activated with glutaraldehyde. It is also possible that whole cells containing the ketoredeuctase enzyme or both the ketoreductase enzyme and a recycling system may be immobilized, if desired, in the practice of this invention. Various exemplary methods for immobilization of both whole cells and enzymes which may be used in the practice of this invention are described in Methods in Enzymology 44 (1976), K. Mosbach editor, Immobilization of Enzymes and Cells, Gordon F. Bickerstaff, ed., Humana Press, Totowa, N.J. (1997) and in Biocatalytic Production of Amino Acids and Derivatives, D. Rozzell and F. Wagner, Eds., Hanser Publishers, Munich, (1992) pp. 279-319, all hereby incorporated by reference. It is understood that other similar methods exist and may also be used in the practice of this invention.
In the next step of the method of this invention, the substituted xcex2-hydroxydiester, which is the immediate product of the diasereoselective reduction of the substituted xcex2-ketodiester, is hydrolyzed regioselectively to the mono-carboxylic acid (FIG. 6). 
As used herein, the term xe2x80x9chydrolyzed regioselectivelyxe2x80x9d refers to the conversion of only one of the two ester groups (either the ester adjacent to the R-group or the ester 2 carbons removed from the R-group) as shown in the structure in FIG. 6 to the substantial exclusion of the other. As it relates to hydrolyzing regioselectively an ester in a molecule containing two or more ester groups, the term xe2x80x9csubstantial exclusionxe2x80x9d means converting at least about 80%, preferably at least about 90%, and more preferably at least about 95% of one ester group while converting less than about 20%, preferably less than about 10%, and more preferably less than about 5% of any other ester group in the molecule. As long as only one of the two ester functional groups is hydrolyzed regioselectively to the substantial exclusion of the other, regardless of which one is hydrolyzed, the two carboxylate groups become chemically distinguishable as the non-hydrolyzed ester and the carboxylic acid.
Regioselective hydrolysis can be achieved enzymatically using a hydrolytic enzyme. Any hydrolytic enzyme capable of regioselective hydrolysis of the substituted xcex2-hydroxydiester to the mono-carboxylic acid may be used. Suitable enzymes for this regioselective hydrolysis include proteases, amidases, lipases, esterases and the like. Many broad range lipases, proteases, and esterases are known that can hydrolyze esters with high regioselectivity. Suitable enzymes for regioselective hydrolysis of a given substituted xcex2-hydroxydiester in accordance with the invention can be identified by routine screening of various hydrolytic enzymes. Examples of such hydrolytic enzymes can be found in the Chirazyme Screening Set or the ICR Screening Set, both available from BioCatalytics Inc. (Pasadena, Calif. USA). In a typical screening experiment, individual reaction mixtures are set up with each of the candidate hydrolytic enzymes and the target substituted xcex2-hydroxydiester to be regioselectively hydrolyzed, and the progress of the reaction is monitored by any convenient assay method. Such assay methods include, but are not limited to, gas chromatography, thin-layer chromatography, high performance liquid chromatography, and the like. It is well known by persons skilled in the art how to identify and select a suitable hydrolytic enzyme for regioselective hydrolysis.
Alternatively, regioselective hydrolysis of the substituted xcex2-hydroxydiester can be accomplished chemically by using an appropriate base under reaction conditions permitting the regioselective hydrolysis of only one of the two ester functional groups to the substantial exclusion of the other. Such regioselective hydrolysis can be achieved using a variety of different bases, including, but not limited to, mineral bases such as sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, and calcium carbonate. Ammonium hydroxide may also be used. Other bases that may be used for regioselective hydrolysis in the practice of this invention include tertiary amine bases such as triethylamine, trimethylamine, tributylamine, tribenzylamine, and the like. The regioselective hydrolysis may be carried out in an aqueous reaction medium, in an aqueous reaction medium containing various amounts of added organic solvent, or in an organic medium containing only small amounts of water. The reaction medium may also be an aqueous/organic two-phase system, if desired. Determination of appropriate conditions for achieving regioselective mono-hydrolysis can be accomplished by routine experimentation by those skilled in the art. It the practice of this invention, it has been found that too high a molar ratio of base:substituted xcex2-hydroxydiester results in over-hydrolysis (that is, hydrolysis of both of one ester functional groups, or at least some of the second ester functional group); whereas too low a molar ratio of base:substituted xcex2-hydroxydiester results in residual amounts of unhydrolyzed diester. For achieving regioselective mono-hydrolysis of the substituted xcex2-hydroxydiester, the preferred molar ratio of base:substituted xcex2-hydroxydiester ranges from about 1:1 to about 1.5:1. The preferred temperature range for the hydrolysis reaction is from about 4xc2x0 C. to about 100xc2x0 C., and more preferably from about 20xc2x0 C. to about 50xc2x0 C. Although aqueous reaction conditions are typically employed for the regioselective mono-hydrolysis of the substituted xcex2-hydroxydiester, co-solvents may be used, if desired, to improve the solubility of the diester or to modulate the rate of the reaction. Suitable co-solvents include ethanol, methanol, isopropanol, tetrahydrofuran, dioxane, dimethylsulfoxide, dimethyl formamide, and the like.
Following the regioselective mono-hydrolysis of the substituted xcex2-hydroxydiester, either the carboxylic acid or the ester functional group is converted to an amine, or derivative thereof, by means of a stereospecific chemical rearrangement. The rearrangement reactions that can be used in the practice of this invention include the Curtius rearrangement and modified versions of the Curtius rearrangement, the Lossen rearrangement and the Hoffmann rearrangement (FIG. 6). These rearrangement reactions are well-studied reactions that are well known to those skilled in the art.
In one embodiment, the carboxylic acid is converted to an amine, or derivative thereof, by means of the Curtius-type rearrangement using the reagent diphenylphosphoryl azide (DPPA). Heating of the mono-carboxylic acid, produced by regioselective mono-hydrolysis of the substituted xcex2-hydroxydiester, with DPPA in the presence of stoichiometric amounts of triethyl amine (TEA) in toluene gives in one step the aminoalcohol rearrangement product as the cyclic carbamate (urethane) derivative (FIG. 7). 
FIG. 7: Rearrangement of the acid with diphosphorylazide and triethyl amine gives a cyclic carbamate as the only product regardless of the presence or absence of alcohol in the reaction mixture. Protection of the alcohol first gives the free amine.
Formation of the mono acid to the corresponding acid azide after treatment with oxalyl chloride and sodium azide, and rearrangement by heating of the azide in tert-butanol or ethanol gave the cyclic carbamate (FIG. 7) as the major product. Small amounts of the ethyl carbamate can be observed when the reaction is carried out in ethanol. The urethane derivatives can be further converted to the corresponding amines, if desired, by hydrolysis under either acidic or basic conditions. The hydroxy group may be protected, if desired, prior to rearrangement. Typical protecting groups include, but are not limited to, simple esters such as acetyl, butyryl, benzoyl, phenylacetyl, and the like; ethers such as methyl ethoxymethyl, dihydropyranyl, and the like; silyl ethers such as t-butyl dimethyl silyl, trimethyl silyl, and the like; and carbonate esters such as t-butyloxycarbonyl, carbobenzyloxy, and the like.
In another embodiment of this invention, the non-hydrolyzed ester functional group is converted to an amine. This conversion is accomplished in two steps. First, the ester is reacted with ammonia, hydrazine, or hydroxylamine to form the corresponding carboxamide, hydrazide, or hydroxamic acid, respectively. Then, the carboxamide, hydrazide, or hydroxamic acid is subjected to conditions permitting the stereospecific rearrangement to form the amine, or a derivative thereof. In each case, the rearrangement proceeds with retention of configuration of the migrating atom, and as a result, the optical purity of the final product is dependent on the stereoselectivity of the enzymatic reduction.
The stereospecific rearrangement may be carried out on the carboxamide via the Hofmann-type rearrangement [E. S. Wallis and J. F. Lane, Organic Reactions III, 267 (1949) and references therein; P. A. S. Smith, Trans. N.Y. Acad. Sci. 31, 504 (1969) and references therein; S. Simons, J. Org Chem. 38, 414 91973) and references therein; W. L. F. Armarego et al, J. Chem. Soc. Perkin Trans. I, 2229 (1976) and references therein; all hereby incorporated by reference]; on the hydroxamic acid via the Lossen rearrangement [S. Bittner et al (Tet. Lett. 23, 1965-8 (1974) and references therein; L. Bauer and O. Exner, Angew. Chem. Int. Ed. 13, 376 (1974) and references therein; all hereby incorporated by reference]; or on the hydrazide via the Curtius rearrangement [Yamada, S. Chem. Pharm. Bull. 22, 1398 (1974); P. A. S. Smith, Organic Reactions III, 337 (1946) and references therein; J. H. Saunders and R. J. Slocombe, Chem. Rev. 43, 205 (1948) and references therein; D. V. Banthorpe in The Chemistry of the Azido Group, S. Patai Ed., Interscience, New York, 1971, pp. 397-405 and references therein; J. D. Warren and J. D. Press, Synth. Comm. 10, 107 (1980) and references therein; all hereby incorporated by reference].
The invention will now be further described by the following examples, which are presented here for illustrative purposes only and are not intended to limit the scope of the invention.